Essential oil constituents, anticancer and antimicrobial activity of Ficus ...

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American Journal of Essential Oils and Natural Products 2016; 4(1): 01-06

ISSN: 2321 9114 AJEONP 2016; 4(1): 01-06 © 2016 AkiNik Publications Received: 15-11-2015 Accepted: 18-12-2015 Emmanuel E Essien Department of Chemistry, University of Uyo, Akwa Ibom State, Nigeria. Jennifer M Newby Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA. Tameka M Walker Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA. Isiaka A Ogunwande, Department of Chemistry, Lagos State University, Badagry Expressway, Lagos State, Nigeria. William N Setzer Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA. Olusegun Ekundayo Department of Chemistry, University of Ibadan Ibadan, Oyo State, Nigeria.

Correspondence: Emmanuel E Essien Department of Chemistry, University of Uyo, Akwa Ibom State, Nigeria.

Essential oil constituents, anticancer and antimicrobial activity of Ficus mucoso and Casuarina equisetifolia leaves Emmanuel E Essien, Jennifer M Newby, Tameka M Walker, Isiaka A Ogunwande, William N Setzer, Olusegun Ekundayo Abstract Essential oils were obtained by hydrodistillation of Ficus mucoso Welw. and Casuarina equisetifolia L. leaves and analyzed for their constituents by means of gas chromatography-mass spectrometry (GC-MS). The major volatile components of F. mucoso leaves were -phellandrene (13.0%), p-cymene (11.3%), germacrene D (10.5%), -caryophyllene (9.7%), 1,8-cineole (9.5%) and -copaene (8.7%). High levels of -phellandrene (40.6%), p-cymene (15.7%), 1,8-cineole (14.1%) and terpinolene (8.4%) characterized C. equisetifolia oil. Ficus mucoso oil demonstrated strong inhibitory activity against Hs578T human breast tumor cells (98.18%) relative to PC-3 human prostate carcinoma cells (47.17%); F. mucoso and C. equisetifolia essential oils also exhibited significant antimicrobial activity (39-625 μg/mL) on pathogens employed in the assays. Keywords: Ficus mucoso, Casuarina equisetifolia, essential oils composition, cytotoxicity, antimicrobial activity

1. Introduction Aromatic plants are frequently used in traditional medicine because of their essential oils. In the last few years, there has been an increase in the use of aromatic medicinal plants and their essential oils in scientific research and industrial applications including nutritional, pharmaceutical, and cosmetic uses [1–4]. At present, approximately 3000 essential oils are known, 300 of which are commercially important especially for the pharmaceutical, agronomic, food, cosmetic, and perfume industry [5]. Moreover, various potent biological activities including antimicrobial, antioxidant, anti-inflammatory, and anticancer are attributed to essential oils [6–7]. Ficus mucoso Welw. is a large tree 30-40 m high with a spreading open crown, in the rain forest, often on river banks. It belongs to the family Moraceae [8]. The young leaves and fruits are edible and have a pleasing aroma. The plant is used in the treatment of insanity, generalized edemas and leprosy. The bark and leaf decoction is taken for diarrhea and dysmenorrhea [9]. In several parts of the world, Ficus species are used in traditional medicines. Ficus thonningii, F. natalensis, F. exasperata, F. leprieurii and F. asperifolia have been implicated for use in venereal diseases, urinary tract infections, viral infections and as antiseptic [10]. There is a paucity of information on the phytochemistry of F. mucoso and biological activity of the essential oil, however a number of researchers have reported on the leaf volatile constituents of some Ficus species [11-16]. Casuarina equisetifolia L. belongs to the plant family Casuarinaceae. It is a rapid- growing tree to 25 m high, native of Australia and occurring commonly in the West African region. A number of forms exist, some with drooping, some with horizontal to erect branches [17]. The leaf is used in the treatment of toothache and dysentery [10]. The plant is a source of biologically active compounds such as catechin, ellagic acid, gallic acid, quercetin and lupeol, which are antioxidants [18], coumaroyl triterpenes [19] and d-gallocatechin (casuarina) [20]. The plant is also known to store tannins [21] and proline [22]. Casuarina equisetifolia has been reputed to demonstrate antimicrobial activity [23]; the seed and fruit contain essential oils [14]. There have been sustained increases in the incidences of breast and prostate cancer in developing countries in recent years and intensive scientific research on this subject matter is imperative. Sixty percent of currently used anticancer drugs are derived one way or the other from natural sources [24-25]. ~ 1 ~ 

 

American Journal of Essential Oils and Natural Products

  As a part of our phytochemical and biological studies on natural essential oils, the aim of this work was to investigate the chemical composition of the leaf essential oils of F. mucoso and C. equisetifolia and their potential antimicrobial and antiproliferative activities on target pathogens and human breast and prostate cancer cell lines, respectively. This is the first report on the biological activity of the essential oils from these plants. 2. Materials and methods 2.1 Plants collection and authentication The mature fresh leaves of F. mucoso and C. equisetifolia were collected in the month of July within the University of Ibadan, Nigeria. Plant samples were authenticated by Mr. F. Usang of the Herbarium Headquarters, Forest Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where voucher specimens (FHI 407412 and FHI 107411, respectively) were deposited. The plant materials were air-dried under a laboratory shade prior to extraction. 2.2 Extraction of the volatile oils Essential oils were obtained by hydrodistillation of the pulverized air-dried plant samples (500 g) in an all glass Clevenger-type apparatus in accordance with the British Pharmacopoeia specifications [26]. The distillation time was 4 h in all experiments. The oils obtained were stored under refrigeration until moment of analyses. 2.3 Gas chromatography-mass spectrometry analyses (GC/MS) The volatile oils were subjected to GC-MS analyses on an Agilent system consisting of a model 6890 Gas chromatograph, a model 5973 Mass Selective Detector (MSD) and an Agilent ChemStation Data system. The GC Column was an HP-5ms fused silica capillary coated with (5% phenyl)methylpolysiloxane (equivalent to USP phase G27) stationary phase, film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 8.28 psi and flow rate of 1.0 mL/min. The inlet and MSD detector temperatures were maintained at 200 °C and 230 °C, respectively, while the MS transfer-line temperature was 280 °C. The GC oven temperature was programmed as follows: 40 °C initial temperature held for 10 min; increased at 3°/min to 200 °C; increased 2°/min to 220 °C. The samples were dissolved in CH2Cl2 to give a 1% w/v solution and a split injection technique was used. The split ratio was 1:30. Mass spectra were recorded at 70 eV. Identification of each individual constituent of the essential oils was achieved based on their retention indices (determined with reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns (NIST database/ ChemStation data system) [27]. 2.4 Cell culture media Human Hs578T breast ductal carcinoma cells (ATCC. No. HTB-129) [28] were grown in 3% CO2 environment at 37 °C in DMEM with 4500 mg glucose per liter of medium, supplemented with 10% fetal bovine serum, 10 μg bovine insulin, 100,000 units penicillin and 100 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO3, pH 7.35. Human PC-3 prostatic carcinoma cells (ATCC No. CRL-1435) [29] were grown in 3% CO2 environment at 37 °C in RPMI1640 medium with l-glutamine, supplemented with 10% fetal

bovine serum, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 15 mM Hepes and 23.6 mM NaHCO3. 2.5 Cytotoxicity screening Hs578T cells were plated into 96-well cell culture plates at 1.0×105 cells per well and PC-3 cells at 1.9 × 104 cells per well. The volume in each well was 100 μL for both cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 μL growth medium containing 2.5 or 1.0 μL of DMSO solution of oil (1% w/w in DMSO), giving a final concentration of 250 or 100 μg/mL respectively for each oil. Hs 578T cells were tested at final concentration at 250 μg/mL and PC-3 cells at final concentration of 100 μg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 and 10 μL DMSO/mL) were used. Tingenone (250 and 100 μg/mL) was used as a positive control [30] . After the addition of compounds, plates were incubated for 48 h at 37 °C. The medium was then removed by suction and 100 μL of fresh medium was added to each well. In order to establish percent kill rates, the cell Titer 96® AQueous NonRadioactive Cell Proliferation Assay was performed [31]. After colorimetric readings were recorded in triplicates (using a molecular Devices SpectraMAX Plus microplate reader, 490 nm), average absorbances, standard deviations, and percent kill ratios (% killoil/% killDMSO) were calculated. 2.6 Antimicrobial screening Essential oils were screened for antibacterial activity against the Gram-positive bacteria, Bacillus cereus (ATCC No. 14579) and Staphylococcus aureus (ATCC No 29213) and the Gram negative bacteria, Pseudomonas aeruginosa (ATCC No 27853) and Escherichia coli (ATCC No 254922). Minimum inhibitory concentration (MIC) was determined using microbroth dilution technique [32]. Dilutions of the oils were prepared in cation-adjusted Mueller Hinton Broth (CAMHB) beginning with 50 μL of 1% w/w solutions of essential oils in DMSO plus 50 μL CAMHB. The oil solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 × 108 colony forming units (CFU)/mL were added to each well. Plates were incubated at 37 °C for 24 h; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive control while DMSO was used as a negative control. Antifungal activity was determined as described above using Candida albicans (ATCC No. 10231) in yeast nitrogen base growth medium with approximately 7.5 × 107 CFU/mL. Antifungal activity against Aspergillus niger (ATCC No 16401) was determined as above using potato dextrose broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control. Both C. albicans and A. niger were maintained at 32 °C and 25 °C respectively for 24 h. 3. Results & Discussion The identified leaf volatiles of F. mucoso are listed in Table 1. A total of thirty five constituents were identified which represent 100% of the leaf essential oil. It was found that the terpenoid content of the leaf is high (89.6%), primarily four monoterpene hydrocarbons accounted for 29.7% while fourteen sesquiterpene hydrocarbons made up 39.9% of the volatile oil. Seven oxygenated monoterpenoids constituted 16.1% and five oxygenated sesquiterpenoids accounted for

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American Journal of Essential Oils and Natural Products

  3.9% of the leaf oil. The aromatic compounds represented by methyl salicylate, dill apiole and (3Z)-hexenyl benzoate constituted 4.1% of the oil; two aliphatic oxygenated compounds were identified in 6.4% concentration. The main constituents in this oil were -phellandrene (13.0%), pcymene (11.3%), germacrene D (10.5%), -caryophyllene (9.7%), 1,8-cineole (9.5%), -copaene (8.7%) and terpinolene (4.9%). (E)-3-methyl-non-2-en-4-one and two oxygenated sesquiterpenoids, spathulenol and carotol, were detected as components of F. mucoso leaf essential oil. Previous studies on Nigerian grown Ficus species have revealed the abundance of 1,8-cineole (13.8%), (E)-phytol (13.7%) and p-cymene (11.4%) in Ficus exasperata [11]; acorenone (20.7%) and phytol (16.2%) in Ficus lutea, with Ficus polita consisting mainly of phytol (23.3%) and 6,10,14-trimethyl-2pentadecanone (15.0%), and Ficus thonningii rich in 6,10,14trimethyl-2-pentadecanone (18.8%) and phytol (14.7%) [12]; βcaryophyllene (37.0%), ethyl octanoate (14.9%) and methyl octanoate (8.3%) in Ficus mucosa [13]; 6,10,14-trimethyl-2pentadecanone (25.9%), geranyl acetone (9.9%), heneicosane (8.4%) and 1,8-cineole (8.2%) in F. elastica [14]; α-cadinol (25.1%), germacrene-D-4-ol (14.9%), γ-cadinene (11.8%) and α-muurolene (9.6%) in F. benghalensis [15]. Both 6,10,14trimethyl-2-pentadecanone and phytol have been described as marker components of the oils of Nigerian grown Ficus species [12]. Some major constituents of previous studies on the essential oil of Ficus plants from Nigeria such as acorenone B, (E)phytol, 6,10,14-trimethyl-2-pentadecanone, hexadecanoic acid, ethyl octanoate, tricosane, hexacosane and cyclotetradecane were not identified in the F. mucoso leaf oil. Adebayo et al. [15] also reported similar variation of F. benghalensis oil composition from previous published data. This observed variation in compositional profile may be attributed to chemotype as well as climatic and ecological conditions in the place of collection. Chemical analyses of C. equisetifolia leaf essential oil revealed the occurrence of twenty five components accounting for 100% of the oil. Identified compounds characterized in the oil and their percentage concentrations are shown in Table 2. The oil was comprised of monoterpenoids, C13-norterpenoids, sesquiterpenoids, aromatic and simple aliphatic compounds. Moderate amounts of alcohols (17.9%), ketones (3.4%), aldehydes (1.3%), aromatic esters and ether (4.4%) were identified in the oil. The monocyclic compounds of the pcymene group and bicyclic compounds of the camphene group were present in the monoterpenoid class. In the p-cymene group, –phellandrene, p-cymene, terpinolene and the oxides, 1,8-cineole and –terpineol were representative compounds while in the camphene group, -pinene and β-pinene were detected. The C13-norterpenoids identified were (E)-βdamascenone, geranyl acetone and (E)-β-ionone. It was found that the terpenoid content of the leaf oil was high (94.7%). Five monoterpene hydrocarbons (69.2%) made up the bulk of the oil with their oxygenated derivatives (18.4%). Six sesquiterpene hydrocarbons (3.8%), three oxygenated sesquiterpenes (3.3%), four aromatic compounds (4.4%) and one oxygenated aliphatic compound (0.9%) were also classified in the oil. The aromatic ether and esters detected

were methyl salicylate, (3Z)-hexenyl benzoate, hexyl benzoate and dill apiole, which occurred as minor components of the leaf oil. The quantitatively significant compounds were phellandrene (40.6%), p-cymene (15.7%), 1,8-cineole (14.1%) and terpinolene (8.4%). Ogunwande et al. [14] reported that the major compounds in C. equisetifolia leaf essential oil were pentadecanal (32.0%) and 1,8-cineole (13.1%); significant quantities of α-phellandrene (7.0%), apiole (7.2%) and αterpinene (6.9%) were present. The main constituents of the fruit were caryophyllene-oxide (11.7%), trans-linalool oxide (11.5%), 1,8-cineole (9.7%), α-terpineol (8.8%) and α-pinene (8.5%). In vitro cytotoxic assays carried out in order to evaluate the biological potentials of the investigated oils revealed potent inhibitory effects of F. mucoso leaf essential oil against Hs578T cells (98.18% kill at 250 μg/mL), however no significant effect on PC-3 cells (Table 3). Casuarina equisetifolia oil showed no cytotoxic activity on either of the cell lines. It is well known that the biological activities of an essential oil may depend on the main constituents or a synergy between the major and some minor compounds [33]. The chemical profiles of both leaf oils are qualitatively and quantitatively distinct (Table 1 and 2). Major sesquiterpenes constituents in F. mucoso oil, such as α-copaene, βcaryophyllene and germacrene D, were either not detected or found in trace amount in C. equisetifolia oil, have shown cytotoxic activity in previous studies [33-35]; however, αphellandrene, p-cymene, 1,8-cineole and terpinolene were common to both oils. Hence, the compositional pattern of F. mucoso leaf oil may have influenced such notable antiproliferative effect against the Hs578T cell line. α-Copaene, a tricyclic sesquiterpene, exhibited cytotoxic effects on MCF-7 breast carcinoma [33] and N2a-NB neuroblastoma [36] cell lines; β-caryophyllene has been described as a potent cytotoxic compound over a wide range of cell lines [33-37]; Salvador et al. [38] attributed the potent cytotoxic activity of Casearia lasiophylla leaf essential oil against several cell lines to a high content of germacrene D and β-caryophyllene; and germacrene D has shown cytotoxic activity to both Hs578T and PC-3 cell lines [34]. Potent cytotoxic activities were considered at ≥90% lethality at tested concentrations of 250 and 100 ppm for both breast and prostate carcinoma cells, respectively, in this report. The antibacterial and antifungal activities of the leaf volatile oils of F. mucoso and C. equisetifolia demonstrated broad spectrum antimicrobial activity (Table 4). Casuarina equisetifolia oil exhibited stronger antibacterial effect on B. cereus, S. aureus (39 μg/mL) and E. coli (312 μg/mL) compared with F. mucoso oil. Candida albicans was more sensitive to C. equisetifolia oil while A. niger showed more susceptibility to F. mucoso volatile oil. It has been documented that Gram-positive bacteria are more sensitive to chemical compounds than Gram-negative bacteria due to the relative thickness of their cell walls [39]. This is obvious by the sensitivity of the Gram-positive bacteria to the tested essential oils in the assay. It should also be noted, however, that prominent constituents common to both oils such as α-  phellandrene, p-cymene, 1,8-cineole and terpinolene, along with other constituents have exhibited antimicrobial activity [40-43] .

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American Journal of Essential Oils and Natural Products

  3.1 Tables Table 1: Chemical constituents of Ficus mucoso leaf essential oil a

RI 870 940 1004 1024 1031 1087 1102 1190 1194 1199 1217 1218 1349 1376 1390 1392 1408 1419 1428 1452 1459 1477 1482 1486 1495 1500 1510 1523 1564 1572 1580 1593 1625 1640 1653

b

RI 875 939 1005 1026 1033 1088 1098 1189 1190 1198 ---1218 1368 1376 1390 1391 1409 1418 1426 1454 1461 1477 1480 1485 1494 1499 1508 1524 1564 1570 1576 1594 1622 1640 1653

Compoundc,d (3Z)-Hexenol α-Pinene α-Phellandrene p-Cymene 1,8-Cineole Terpinolene Linalool α-Terpineol Methyl salicylate Safranal (E)-3-Methyl-non-2-en-4-one β-Cyclocitral α-Cubebene α-Copaene β-Cubebene β-Elemene α-Gurjunene β-Caryophyllene α-Ionone α-Humulene Alloaromadendrene γ-Muurolene Germacrene D β-Ionone Bicyclogermacrene α-Muurolene (E,E)-α-Farnesene δ-Cadinene (E)-Nerolidol (3Z)-Hexenyl benzoate Spathulenol Carotol Dill Apiole τ-Cadinol α-Cadinol

Area (%) 3.4 0.5 13.0 11.3 9.5 4.9 2.1 0.6 1.2 0.7 3.0 1.0 0.6 8.7 0.4 0.3 0.2 9.7 0.6 3.5 2.0 0.2 10.5 1.6 0.5 1.3 0.2 1.8 0.5 1.3 1.7 0.9 1.6 0.3 0.5

QIe (%) 91 91 94 97 98 96 93 92 95 95 98 96 98 99 91 98 93 99 98 99 99 91 96 96 93 98 87 99 94 98 95 96 96 90 95

aRI,

calculated retention indices; bRI, retention index from literature; cOrder of elution on HP-5ms capillary column; dIdentification by comparison of the mass spectral and retention index data; eQI, ‘quality index’, reflects the fit comparison of experimental mass spectrum and NIST library mass spectrum. Table 2: Chemical constituents of Casuarina equisetifolia leaf essential oil a

RI 855 940 977 1003 1023 1031 1087 1190 1194 1218 1385 1392 1418 1455 1460 1480 1486 1510 1541 1564 1570 1577 1593 1626 1723

RIb 854 939 980 1005 1026 1033 1088 1189 1190 1218 1380 1391 1418 1453 1459 1480 1485 1508 1542 1564 1570 1576 1594 1622 1722

Compoundc,d (2E)-Hexenal α-Pinene β-Pinene α-Phellandrene p-Cymene 1,8-Cineole Terpinolene α-Terpineol Methyl salicylate β-Cyclocitral (E)-β-Damascenone β-Elemene β-Caryophyllene Geranyl acetone Aromadendrene Germacrene D (E)-β-Ionone (E,E)-α-Farnesene α-Calacorene (E)-Nerolidol (3Z)-Hexenyl benzoate Hexyl benzoate Carotol Dill apiole (E,E)-Farnesol

aRI,

Area (%) 0.9 3.7 0.8 40.6 15.7 14.1 8.4 0.5 0.5 0.4 0.4 1.0 0.8 0.5 0.8 0.6 2.5 0.3 0.3 1.4 1.1 0.8 1.5 2.0 0.4

QIe (%) 95 95 91 93 97 99 98 94 96 94 78 96 99 91 90 93 96 96 93 99 92 78 89 96 94

calculated retention indices; bRI, retention index from literature; cOrder of elution on HP-5ms capillary column; dIdentification by comparison of the mass spectral and retention index data; eQI, ‘quality index’, reflects the fit comparison of experimental mass spectrum and NIST library mass spectrum ~ 4 ~ 

 

American Journal of Essential Oils and Natural Products

  Table 3: Cytotoxic activity of F. mucoso and C. equisetifolia leaf essential oilsa Hs578Tb PC-3c 98.18 ±1.17 47.17 ±1.63 0 1.81±2.37 100.0 (IC50 = 78.2±8.3 100 (IC50 = 31.3±1.1 μg/mL) [34] μg/mL) [34] 100 (IC50 = 26.0±2.0 100.0 (IC50 = 55.2±5.3 Germacrene D μg/mL) [34] μg/mL) [34] Tingenone 100.0 100.0 a% kill at tested concentrations in triplicate (standard deviations in parentheses); negative controls, medium and DMSO had no effects in the assay; bHuman breast (ductual) tumor cells (% kill at 250 μg/mL); cHuman prostate tumor cells (% kill at 100 μg/mL). Reference compound; tingenone at concentration of 250 or 100 ppm. Sample F. mucoso C. equisetifolia βCaryophyllene

Table 4: Antimicrobial activity of F. mucoso and C. equisetifolia volatile oils (MIC, μg/mL) Sample B.c S.a E.c P.a C.a A.n F. mucoso 625 312 625 625 625 78 C. equisetifolia 39 39 312 625 312 156 Positive control 1.22a 0.61a 2.44a 1.22a 0.61b 0.61b B.c., Bacillus cereus (ATCC No. 14579); S.a, Staphylococcus aureus (ATCC No 29213); E.c, Escherichia coli (ATCC No. 25922); P.a, Pseudomonas aeruginosa (ATCC No. 27853); C.a, Candida albicans (ATCC No. 10231); A.n, Aspergillus niger (ATCC No. 16401). aGentamicin sulphate; bAmphotericin B; Negative control, DMSO had zero effect in the assay.

4. Conclusions The biological activities of F. mucoso and C. equisetifolia essential oils are a function of their distinct chemical profiles. This study indicates that F. mucoso leaf oil possesses strong cytotoxic effects against Hs578T carcinoma cells. Furthermore, F. mucoso and C. equisetifolia oils demonstrated significant broad spectrum in-vitro antimicrobial activity against medicinally important pathogens in the assay. The present findings would provide further insight on the plants therapeutic applications and enhance the exploitation of these oils for other biological purposes. 5. References 1. Kebede A, Hayelom M. The design and manufacturing of essential oil distillation plant for rural poverty alleviation in Ethiopia. Ethiopian Journal of Environmental Studies and Management. 2008; 1(1):84-91. 2. Ramadan MF. Nutritional value and applications of Nigella sativa essential oil: a mini review. Journal of Essential Oil Research. 2015; 27(4):271-275. 3. Edris AE. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: a review. Phytotherapy Research 2007; 21:308-323. 4. Kejlová K, Jírová D, Bendová H, Gajdoš P, Kolářová H. Phototoxicity of essential oils intended for cosmetic use. Toxicology in vitro 2010; 24(8):2084-2089. 5. Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils – a review. Food Chemical Toxicology 2008; 46:446-475. 6. Essien EE, Walker TM, Ogunwande IA, Bansal A, Setzer WN, Ekundayo O. Essential oil composition, cytotoxicity and antimicrobial activities of Datura metel L. from Nigeria. International Journal of Essential Oil Therapeutics. 2010; 4:69-72. 7. Bayala B, Bassole HN, Scifo R, Gnoula C, Morel L, Lobaccaro JA et al. Anticancer activity of essential oils

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